LAUSR

Background: Colorectal cancer and Mammary analogue secretory carcinoma harboring Neurotrophic tyrosine receptor kinase (NTRK/TRK) gene rearrangements developed resistance to Entrectinib, ALK/ROS/TRK inhibitor. We previously demonstrated that the next generation pan-TRK inhibitor,ONO-5390556 may potently overcome Entrectinib-resistance mutations. Furthermore, inhibition of Trk phosphorylation in tumors has excellent correlation with the in vivo anti-tumor effect by ONO-5390556 (AACR2016, Kozaki et al). Purpose: Accurate measurement of target inhibition in a phase 1 clinical trial is critical to informing selection of appropriate doses for ONO-5390556 in more advanced clinical trials. Plasma inhibitory activity (PIA) assay has recently been performed as a surrogate assay in clinical trials of FLT3, MET and ALK kinase inhibitors. The assay employs the incubation of Tyrosine Kinase expressing cell lines in aliquots of plasma collected at various time points from patients treated with TKI. Herein, we report the preclinical evaluation of PIA assay in accordance with an inhibition of TRK phosphorylation in tumors. Methods: KM12 cells, human colorectal cancer cell lines expressing TPM3-TRKA, were implanted subcutaneously into nude mice. Mice were randomized when the mean tumor volume was 150-600 mm3. Tumors and blood were collected from mice, 2, 4, 7 and 24 hours after the single treatment of ONO-5390556 with the doses of 0.06 and 0.6 mg/kg. The collected tumors were disrupted and phosphorylated TrkA in tumors was detected by Electrochemiluminescence (ECL). In a PIA assay, KM12 was incubated in plasma from the collected blood for 4 hours and phosphorylated TrkA in the cells was detected by ECL.. Results: Treatment with ONO-5390556 at doses of 0.06, 0.6 mg/kg resulted in a significant inhibition of Trk phosphorylation in tumors up to 24 hours. Compared to inhibition of P-TRK in tumors, the inhibition of P-TRK in PIA sustained until 7 hours but rapidly decreased at 24 hours after administration. The levels of both tumors and PIA showed in a dose-dependent inhibition and an excellent correlation until 7 hours. Inhibition of TRK phosphorylation (PIA/Tumor, %)0.06 mg/kg (2,4,7,24h) : 56.3/41.4 (PIA/Tumor, %), 51.9/53.5, 43.0/66.5, -5.5/51.30.6 mg/kg (2,4,7,24h) : 90.2/84.3, 88.4/93.0, 87.4/95.7, 8.3/83.3Conclusion: We have validated the PIA assay that correlates with the inhibition of P-TRK in tumors. Our results demonstrated that the potential utility of PIA as a PD marker may contribute to determining the effective dose of ONO-5390556 in the clinical development. Citation Format: Ryohei Kozaki, Ryu Fujikawa, Hikaru Kato, Natsuka Goto, Toshio Yoshizawa. Plasma inhibitory activity (PIA) is a possible pharmacodynamic marker for clinical development of a next generation pan-TRK inhibitor, ONO-5390556 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3727. doi:10.1158/1538-7445.AM2017-3727